Abstract

KatA is the major catalase required for hydrogen peroxide (H2O2) resistance and acute virulence in Pseudomonas aeruginosa PA14, whose transcription is driven from the promoter (katAp1) located at 155 nucleotide (nt) upstream of the start codon. Here, we identified another promoter (katAp2), the +1 of which was mapped at the 51 nt upstream of the start codon, which was responsible for the basal transcription during the planktonic culture and down-regulated upon H2O2 treatment under the control by the master regulator of anaerobiosis, Anr. To dissect the roles of the dual promoters in conditions involving KatA, we created the promoter mutants for each -10 box (p1m, p2m, and p1p2m) and found that katAp1 is required for the function of KatA in the logarithmic growth phase during the planktonic culture as well as in acute virulence, whereas katAp2 is required for the function of KatA in the stationary phase as well as in the prolonged biofilm culture. This dismantling of the dual promoters of katA sheds light on the roles of KatA in stress resistance in both proliferative and growth-restrictive conditions and thus provides an insight into the regulatory impacts of the major catalase on the survival strategies of P. aeruginosa.

Figures

Figure 1. Promoter region of the katA…

Figure 1. Promoter region of the katA gene.

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( A ) High-resolution S1 nuclease mapping.…

Figure 1. Promoter region of the katA gene.

(A) High-resolution S1 nuclease mapping. The 5′ end of the katA mRNA transcript was determined by high-resolution S1 nuclease mapping. Total RNA (50 μg) that had been prepared from the cells with (+) or without (−) 1 mM H2O2 treatment for 10 min at OD600 of 1.0 in LB were subjected to S1 nuclease mapping. The sequencing reactions (lanes C, T, A and G) were carried out as described in Methods. The two transcriptional start sites of the katA gene are indicated by open (katAp1) and closed (katAp2) arrows. The detailed transcriptional start site of katAp2 promoter is shown on the left by solid line, and its putative −10 box is enclosed in box. (B) Promoter elements of the katA gene. The sequence of the katA promoter region of the katA gene is shown, with the two 5′ ends of katAp1 and katAp2 promoters indicated by the bent arrows. The Anr binding consensus (TTGac-N4-gtCAA) is designated, whose center is located at −29 position from the katAp2 promoter. The putative −10 and −35 boxes are underlined. The N-terminal amino acid sequences of the katA gene are designated.

Figure 2. Transcription profiles of katA promoters…

Figure 2. Transcription profiles of katA promoters in mutants.

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The transcription patterns were assessed by…

Figure 2. Transcription profiles of katA promoters in mutants.

The transcription patterns were assessed by low-resolution S1 nuclease protection assay with H2O2 treatments in oxyR and anr mutants grown in LB (A) and in nirS and norB mutants grown in LB amended with 15 mM KNO3 (B). Total RNA (50 μg) that had been prepared from the wild type (WT) and the mutant (oxyR, anr, nirS, and norB) cells with (+) or without (−) 1 mM H2O2 treatment for 10 min at OD600 of 0.5 were subjected to S1 nuclease assay. The two transcriptional start sites of the katA gene are indicated by open (katAp1) and closed (katAp2) arrows.

Figure 3. Creation of the katA promoter…

Figure 3. Creation of the katA promoter mutants.

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( A ) Schematic representation. The katA…

Figure 3. Creation of the katA promoter mutants.

(A) Schematic representation. The katA gene and its potential promoter elements (−35 and −10 boxes) are designated. The sequences of each −10 box are indicated above the boxes. The katA promoter mutations were constructed by substituting the −10 box with the KpnI site (GGTACC) for the katAp1 mutant (p1m) or the BamHI site (GGATCC) for the katAp2 mutant (p2m) as indicated with the mutated nucleotides underlined. A double mutant for both promoters (p1p2m) was generated as well. (B) Transcription upon H2O2 induction. Total RNA (50 μg) isolated from the wild type (WT) and the katA promoter mutant (p1m, p2m, and p1p2m) cells with (+) or without (−) 1 mM H2O2 treatment for 10 min at OD600 of 0.5 in LB were subjected to low-resolution S1 nuclease assay. The two transcriptional start sites of the katA gene are indicated by open (katAp1) and closed (katAp2) arrows. (C) Catalase activity staining upon H2O2 induction. Catalase activities in cell extracts of the wild type (WT) and the katA null and promoter mutant (katA, p1m, p2m, and p1p2m) cells with (+) or without (−) 1 mM H2O2 treatment for 10 min as in (B) were visualized using 50 μg of proteins in each cell extract. The two catalase bands are indicated by open (KatB) and closed (KatA) arrows. (D) Promoter activity during aerobic planktonic growth. The wild type cells harboring one of the promoter transcription fusions (•, WT; ◇, p1m; □, p2m; ∆, p1p2m) were grown in LB amended with 15 mM KNO3. Culture aliquots were taken at every 2 h from 2 to 10 h post-inoculation and then subjected to β-galactosidase (LacZ) assay. The data represent the average of the means of three independent experiments (two cultures per experiment), with the error bars representing the standard deviations.

Figure 4. Stress resistance of the katA…

Figure 4. Stress resistance of the katA promoter mutants.

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( A , B ) Stress…

Figure 4. Stress resistance of the katA promoter mutants.

(A,B) Stress resistance in planktonic culture. Susceptibility to H2O2 and acidified NaNO2 (aNO2) were assessed for the wild type (WT) and the katA null and promoter mutant (katA, p1m, p2m, and p1p2m) cells that had been cultured for 3 h or for 6 h in LB broth (pH 6.5), and treated for 24 h with either 15 mM H2O2 and 200 μM NaNO2 or in LB broth (pH 6.5) (−). Ten-fold serial cell dilutions from the 3-h (A; logarithmic) and 6-h (B; stationary) cultures were spotted on LB agar plate. The numbers indicate the log (cfu) of the applied bacterial spots. (C) Stress resistance in static culture. The wild type (WT) and the katA null and promoter mutant (katA, p1m, p2m, and p1p2m) cells were statically and anaerobically cultured for 7 days in LB broth (pH 6.5) treated for 1 h with either 100 mM H2O2 and 1.2 M NaNO2 or in LB broth (pH 6.5) (−). Ten-fold serial cell dilutions from the cultures were spotted on LB agar plate. The numbers indicate the log(cfu) of the applied bacterial spots.

Figure 5. Acute virulence of the katA…

Figure 5. Acute virulence of the katA promoter mutants.

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Virulence in Drosophila ( A )…

Figure 5. Acute virulence of the katA promoter mutants.

Virulence in Drosophila (A) and mouse peritoneal (B) infection models. The wild type (WT) and the mutant (katA, p1m, p2m, and p1p2m) cells were prepared and the survival of infected animals was determined as described in Methods. The values are the averages from five replicate experiments for Drosophila (A) and three for mouse (B) infections. Statistical significance based on the log-rank test is indicated (**p < 0.005).

Figure 6. Accumulation of NO 2 −…

Figure 6. Accumulation of NO 2 − in the katA promoter mutants.

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Steady-state level of extracellular NO…

Figure 6. Accumulation of NO2− in the katA promoter mutants.

Steady-state level of extracellular NO2− was measured in the wild type (WT) and the katA null and promoter mutant (katA, p1m, p2m, and p1p2m) cells as well as anr mutant cells that had been grown to OD600 of 0.7. The amount (μM) of NO2− is calculated using the standard curve (r2 = 0.999) and the average values measured from the three independent experiments, with the error bars representing the one positive value of the standard deviations. Statistical significance based on the Student’s t-test is indicated (*p < 0.01; **p < 0.005).